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Revision on the effects of an entomopathogen bacteria Bacillus thuringiensis on the immune response of Red Palm Weevil (Rhynchophorus ferrugineus), is it a question of gender?
Full text link1 Università degli Studi di Palermo, Italia; 2 Università degli Studi dell’Insubria, Italia
Il punteruolo rosso, Rhynchophorus ferrugineus (Olivier 1790) (Coleoptera: Curculionidae) fitofago infestante delle palme e Galleria mellonella L. (Lepidoptera: Pyralidae) parassita delle arnie, sono stati utilizzati come modelli per approfondire le conoscenze della relazione ospite-nematode entomopatogeno.
Gli effetti del nematode entomopatogeno Steinernema carpocapsae (Nematoda: Steinernematidae) sull'immuno attività delle larve di R. ferrugineus e di G. mellonella, sono stati messi a confronto. E’ stata anche valutata la capacità di completare il ciclo vitale di S. carpocapsae nei due insetti.
Sebbene S. carpocapsae sia efficace nel controllo biologico del punteruolo rosso, non è tuttavia in grado di completare il suo ciclo all’interno dell’ospite, mentre lo stesso nematode si riproduce con successo in G. mellonella.
Sulla base di precedenti dati di laboratorio e sulla revisione sistematica della letteratura, gli effetti indotti dal nematode sulla risposta immunitaria del Coleottero e del Lepidottero, sono stati comparati. In particolare sono stati analizzati: la popolazione emocitaria immunocompetente (numero e tipi cellulari), l’attività del sistema profenolossidasi-fenolossidasi (proPO) e infine è stata valutata l’inibizione e proliferazione del batterio simbionte Gram negativo Xenorhabdus nematophila (Enterobacteraceae).Since their discovery, bioinsecticides based on Bacillus thuringiensis (Bt) have demonstrated targeted insect control with minimal environmental impact. Understanding the biology of insect pests, as well as interac- tions with pathogens and immune systems, can help to enhance their effect within these interactions.
On the basis of previous laboratory data and a systematic literature review we analyzed the effects induced by the entomopathogen bacteria Bt, on red palm weevil (RPW), an highly invasive pest of palms that had a significant economic, environmental and social impact when introduced into Italy. The bacteria-induced effects on physiology, immune response and stress answers in the RPW. Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae) were considered. Previously a Bt strain, commercialized against another family of Coleopters, was found to be a potential pathogen of RPW. Data showed that Bt interacts negatively with the growth and the vitality of RPW moreover with hemocytes of R. ferrugineus whose numbers decreased drastically in the hemolymph both in total number and in type. In particular, we focused on the Bt stress- induced infections considering the genders and the instars. The interaction between Bt and RPW hemocytes was analyzed by evaluating the expression heat shock proteins (HSPs) particularly HSP70 in the supernatant of the hemocyte lysate obtained from larvae and adults. HSPs are rapidly synthesized in the cells after stress exposition including infection by pathogens. The western blot analysis, showed that the HSP70 expression was modulated in the time (3h, 6h, 12h, 19, 24h) in the response to Bt treatment, highlighting that Bt is a stress factor for the larvae but also for adults. The protein expression was increased approximately seven times after 3 hours from treatment and after 6 hours it returning to control value. Data show difference in how males and females invest in Bt infection answer. Our data on immunity support the theory that males choose investment in reproduction over investment in immunity. In fact, B. thuringiensis-induced mortality data show the effect that females mount a stronger immune response and they also suffer lower mortality
Does the immune answer to Bacillus thuringiensis infection is the same in larvae, females and males of Rhynchophorus ferrugineus?
No full textBacillus thuringiensis (Bt) è considerato un potenziale batterio entomopatogeno per la lotta al
Rhynchophorus ferrugineus, coleottero da quarantena infestante le palme. In questo lavoro si considerano gli effetti dell’infezione di Bt in larve, maschi e femmine del punteruolo rosso. La patogenicità è stata valutata stimando la LD50 (Lethal Median Dose) e la LT50 (Lethal Median Time), il numero totale di emociti ed il tipo di emociti (Differential Haemocytes counts) ed inoltre l’espressione delle proteine Heat Shock Protein 70 (HSP70) negli emociti e nel cerebro. La mortalità di entrambi i sessi e delle larve aumentava all’aumentare della dose e del tempo di esposizione. Tuttavia le larve hanno mostrato una maggiore mortalità, quasi doppia di quella degli adulti, mentre i valori di LT50 erano superiori nelle femmine rispetto ai maschi e alle larve. Trattamenti con dosi sub letali di Bt inducevano un decremento nel numero di emociti sia per larve che per gli adulti. Nelle larve, tuttavia, vi era un incremento dei plasmatociti e un decremento di enociti e sferulociti. Nelle femmine invece diminuivano tutti i tipi di emociti mentre nei maschi diminuiva il numero di plasmatociti ed incrementava quello dei granulociti. La risposta fisiologica alla infezione da Bt è stata valutata anche mediante la modulazione delle HSP70 negli emociti, mostrando che già dopo 3 ore
dall’ingestione di Bt, si poteva notare un incremento delle stesse. Nel cerebro il massimo picco di
modulazione delle HSP70 si è registrato a 6 ore dall’ingestione. I risultati confermano che il Bt induce uno stato di patogenesi nel punteruolo rosso sia negli adulti che nelle larve già a poche ore dall’ingestione tuttavia gli effetti sono diversi come livello e tipo di risposta.Bacillus thuringiensisis considered a potential useful entomopathogen of Rhynchophorus ferrugineus(RPW), a quarantine pest that attacks the palm trees. We compare the effects of Bt in mature larvae, females and males. The pathogenicity activity of Bt was evaluated estimating: Median Lethal Dose (LD50), Median Lethal Time (LT50), and Total Haemocytes Count (THC) and Differential Haemocytes count (DHC), and finally the expression of the stress protein Heat Shock Protein 70 in the haemocytes and in the brains. RPW mortality exhibited a positive trend with the dosage and duration of exposure to Bt. The larvae were more susceptible than adults and the LD50 of females was even the double value of the one of larvae. Similarly, the LT50 value was higher for females than males and larvae. Treatment with sub-lethal dose of Bt induces a decrement in THC in larvae, females, and males. In treated larvae plasmatocytes decrease while oenocytes and spherulocytes increase. In treated females all type of haemocytes decrease, while in males the number of plasmatocytes decreases and the granulocytes increase. We also registered the stress answer directly on hemocytes, showing that already at 3, hours after eating Bt, it modulates the expression of the stress protein Hsp 70. This effect was also observed in the brain tissue at 6 hours after treatment.
The results confirm that the Bt treatment induces a pathogenic state in RPW larvae and adult of both genders, that answer only after few hours from ingestion, however, the effects are different in magnitude and in type of target. The highlights can be useful to understand better the relationship pathogen-host and to implement the integrated control of phytophagous
AQUATIC SHRIMPS AS A RESOURCES OF BIOACTIVE MOLECULES
No full textTo date, aquatic crayfish play a very important role both environmentally then economically. Depending on the species they can be invasive, bioindicators or used in human nutrition. Among these species today appear to be important one marine and one of freshwater species: respectively Aristaeomorpha foliacea and Procambarus clarkii. The first is an economically important species highly appreciated by consumers, while the second is an invasive species responsible of the disappearance of the autochthonous species and of serious impacts on aquatic ecosystems (SOUTY-GROSSET et al., 2016). In the case of A. foliacea, the production of waste to date is considerable due to the large quantity of exoskeletons that are which constitute both environmental and economic damage for disposal companies. In the case of P. clarkii being that it is a very dangerous species for freshwater ecosystems, to date it is very important carrying out eradication or control of its distribution. Using the exoskeleton of these species is possible to obtain bioactive molecules such as chitosan. All these aspects are part of the purpose of our study which is part of two project: the PO FEAMP 2014-2020 project entitled "Census, characterization, control and valorization of the Louisiana red shrimp (Procambarus clarkii) in Sicily" and Interreg Italia-Malta entitled "Bythos Extend". In particular, the preparations of the flours and the extractions of chitosan and astaxanthin from the exoskeletons have already been developed in the Bythos I project (MAURO et al., 2022). These protocols were used to extract chitosan and astaxanthin from P. clarkii exoskeletons of different Sicilian lakes: the Rosamarina Lake and the Gorghi Tondi Lake. The obtained chitosans were analyzed using a Jasco FT/IR 420 spectrometer. On flours, were evaluated total phenolic content (Folin-Ciocalteu reaction), ferric ion reducing antioxidant power (FRAP) and radical scavenging activity assays (ABTS and DPPH). The reducing power and radical scavenger activity of flour of A. foliacea was significantly higher than P. clarkii. Flours of the two species of P. clarkii, as expected, showed comparable reducing power. ABTS, DPPH and FRAP radical scavenger activity assays were conducted on astaxanthin and chitosan showing no significant differences. These results are important to evaluate a possible use of these bioactive products in cosmetic, nutraceutical, and/or medical application
EFFECT OF AQUEOUS EXTRACTS FROM Posidonia oceanica SEAGRASS ON MOUSE MACROPHAGES AND HUMAN BLOOD BRAIN BARRIER
Full text linkEFFECTS OF ERICARIA BRACHYCARPA EXTRACT ON THE DEVELOPMENT OF ARBACIA LIXULA SEA URCHIN EMBRYOS
No full textBeing macroalgae sessile organisms, they need to constantly adapt to either the abiotic and biotic components of the marine ecosystem and have developed complex adaptations to survive, including the production of bioactive molecules. The aim of this work was to evaluate the effects of an extract of the brown macroalga Ericaria brachycarpa on the development of Arbacia lixula sea urchin embryos, to evaluate the embryotoxic activity from fertilization (0 h) to the pluteus stage (72 h). The range of concentrations tested was chosen to cover a full 0-100 % abnormality curve, with doses ranging from 0 to 40 μg/mL. The extract was added at three developmental endpoints: zygote (0 hpf), gastrula (24 hpf) and pluteus (48 hpf), founding that gastrulae were the most sensitive to the extract with the lowest EC50 (5.366 μg/mL). In all treatments there was a dose-dependent effect. At low concentrations we found a significant variation in two morphometric parameters at the pluteus stage, compared to controls. The highest concentration tested (40 μg/ml) caused 100% mortality of the embryos at all embryo stages. Western Blot experiments showed the modulation of different molecular markers (HSP60, LC3, p62, CHOP and cleaved caspase-7), showing enhanced autophagy at low concentrations and apoptosis at high concentrations. The TUNEL assay confirmed high levels of fragmented DNA in 48 h exposed embryos. Further studies by means of HPLC/MS/QToF are in progress to identify which compound(s) of the E. brachycarpa extract are responsible for the embryotoxic activity
Identification of CPE and GAIT elements in 3’UTR of macrophage migration inhibitory factor (MIF) involved in inflammatory response induced by LPS in Ciona robusta
No full textInnate immune responses face infectious microorganisms by inducing inflammatory responses. Multiple genes
within distinct functional categories are coordinately and temporally regulated by transcriptional ‘on’ and ‘off’
switches that account for the specificity of gene expression in response to external stimuli. Mechanisms that
control transcriptional and post-transcriptional regulation are important in coordinating the initiation and resolution
of inflammation. Macrophage migration inhibitory factor (MIF) is an important cytokine that, in Ciona
robusta, is related to inflammatory response. It is well known that in C. robusta, formerly known as Ciona intestinalis,
the pharynx is involved in the inflammatory reaction induced by lipopolysaccharide (LPS) injection in
the body wall. Using this biological system, we describe the identification of two C. robusta MIFs (CrMIF1 and
CrMIF2). The phylogenetic tree and modeling support a close relationship with vertebrate MIF family members.
CrMIF1 and CrMIF2 possess two evolutionally conserved catalytic sites: a tautomerase and an oxidoreductase
site with a conserved CXXC motif. Real-time PCR analysis shows a prompt expression induced by LPS inoculation
in CrMIF1 and a late upregulation of CrMIF2 and in silico analyses of 3’UTR show a cis-acting GAIT element and a
CPE element in 3’-UTR, which are not present in the 3’-UTR of CrMIF1, suggesting that different transcriptional
and post-transcriptional control mechanisms are involved in the regulation of gene expression of MIF during
inflammatory response in C. robusta
Humoral responses during wound healing in Holothuria tubulosa (Gmelin, 1788)
Full text linkWounds in living organisms trigger tissue-repair mechanisms. The sea cucumber (Holoturia tubulosa) is an excellent model species for achieving a better understanding of the humoral and cellular aspects involved in such healing processes. Consequently, this study assesses data on its morphometric, physiological and humoral responses 1, 2, 6, 24 and 48h after wound induction. In particular, morphometric data on the weight, width, length and coelomic-fluid volume of the species were estimated at different times during our experiments. In addition, the humoral aspects related to the enzymatic activity of esterase, alkaline phosphatase and peroxidase, as well as the cytotoxic activity of cell lysates (CL) and cell-free coelomic fluids (CfCf) are evaluated for the first time. Our results reveal a significant decrease in body length and weight, along with time-dependent, significant changes in the esterase, alkaline phosphatase, peroxidase and cytotoxic activity in both the CL and CfCf. The data obtained lead to the pioneering finding that there is an important time-dependent involvement of morphometric (changes in weight and length) and humoral (enzymatic and cytotoxic) responses in wound healing
Antimicrobial and antibiofilm properties of polypeptide-enriched extracts from the Mediterranean seagrass Posidonia oceanica
No full textDue to the rapid increase of antimicrobial resistance to conventional antibiotics and the diffusion of
multidrug-resistant pathogens worldwide, it is urgent to develop alternative class of therapeutic
molecules. Antimicrobial peptides (AMPs) are considered potential therapeutics in the treatment of
bacterial and fungal infections. We focused on Mediterranean seagrass Posidonia oceanica as a source
of new bioactive molecules with antimicrobial and antibiofilm properties. Polypeptide-enriched
fractions of rhizomes and green leaves of the seagrass were tested against Gram-positive reference
ATCC strains (Staphylococcus aureus, Enterococcus faecalis) and Gram-negative bacteria
(Pseudomonas aeruginosa, Escherichia coli) and towards the yeast Candida albicans. The above
mentioned extracts showed MIC values ranging from 1 .61 μ g/mL t o 7 .5 μ g /mL, against tested
pathogens. Peptide fractions were further analyzed through a high-resolution mass spectrometry and
database search, identifying twelve novel peptides. Some peptides and their derivates were
chemically synthesized and assessed in vitro against bacterial and yeast strains. In particular, two
synthetic peptides, derived from green leaves and rhizomes of P. oceanica, revealed an interestng
antibiofilm activity towards E. coli and P. aeruginosa (BIC50 equal to 17.6 μg/mL and 70.7 μg/mL). In
conclusion, these results support the potential use of discovered peptides as platform for the
development of novel synthetic AMPs with improved pharmaceutical potential against relevant
pathogens
Antimicrobial and antibiofilm properties of polypeptide-enriched extracts from the Mediterranean seagrass Posidonia oceanica
Full text linkENVIRONMENTAL DNA: A POSSIBLE TOOL TO EVALUATE THE VERTEBRATE BIODIVERSITY USING WATER SAMPLES OF THREE SICILIAN LAKE
No full textFreshwater ecosystems play a key role for the biogeochemical cycles and are subject to several anthropic impact that cause a biodiversity loss. Conventional census methods seem to be insufficient and not very convenient for the detection of species. The organisms inhabiting these sites continuously release DNA, called "environmental DNA" (eDNA), through cells, excrement, gametes and/or decomposing material and its evaluation could revolutionize the monitoring of biodiversity (HARPER et al., 2019; SCHENEKAR, 2023). On the other hand, today there are considerable doubts about it: seems to be the non-existence of well-defined protocols which depend on the sampling site, environmental factors and extraction methods; degradation processes are very rapid and the costs of evaluations are very high (many samples are needed in different points). The aim of this study (part of RTDA-PON project) was to fine-tune the sampling and eDNA extraction protocol, reducing the costs and evaluating the potential of this method compared to the conventional methods. Another aim was evaluating the biodiversity of vertebrate in some Sicilian Lake for which until to date no more is known. In this study, the eDNA technique was used for the first time in three Sicilian lakes: Poma Lake, Piana degli Albanesi Lake and Scanzano Lake.The water sampled in each site was filtered and eDNA was extracted using extraction kit. Metabarcoding analysis was performed by IGA Technology Services s.r.l. The results showed the possibility to understand which types of phyla, class, order, family, gender, or species lives in these sites. The species not captured or not sighted using conventional methods were detected thanks to the use of eDNA and vice versa highlighting that the evaluation of eDNA can be useful to evaluate not only aquatic species but also other species that use these sites as a source of supply. Another important result was the possibility to detect DNA of species not typical of freshwater sites highlighting the contamination process. eDNA is a very important tool to evaluate the biodiversity and the health status of the environment, but on the other hand, for a more complete census, it is very important use also other conventional methods at the same times. Our preliminary results showed that eDNA method have the potential to replace the traditional methods of census but it is not yet the time to do it, using both methods is surely the best approach
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